Hello all,
I have a question. I am trying to test my DNA probes (that are biotin labelled) that we have in our lab to perform FISH. Probes are around 80 to 200 bp. I am doing a dot blot, to test our probes. However, I can not see anything when I develop the blot with ECL.
Does anyone have a good protocol that can help me? I feel like, I am missing a fundamental point. It doesn't make sense there is absolutely nothing on the membrane, complete white.
Looking forward to hear ideas,
Dilan
Am I wrong or did you just develop the blot with ECL without frist incubating it with an anti-biotin antibody?
I did such dot blots ages ago using a Streptavidin-AP conjugate and NBT/BCIP as substrate solution.
Hello Sabine,
after I add the probe, I block and incubate with Streptavidin-HRP and use ECL.
I feel like, somehow my DNA probe is not staying on the membrane.
I can't recall that this ever happened to me. I need to check my old (almost ancient) lab books regarding membrane drying and incubation temperatures and times.
May I ask if you remember how did you apply DNA probe to the membrane? Did you use UV and/or denaturation?
Because I think that how I apply the DNA probe might be wrong, so it gets washed away.
Dear Dilan Serdar
As membrane I used nitrocellulose and I denatured the nick-translated DNA probes at 95°C for 5 minutes before spotting them on the nitrocellulose, then I baked it for about 1 hr at 80°C in a drying oven. Hope this helps.
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